The Activity Regulation Of JAK2 And PTPN12 By Vitamin C-MAPK/ERK Signaling Pathway

Vitamin C is an indispensable nutrient in the organism and plays an important role in maintaining the body's homeostasis.A large number of clinical studies have shown that high-dose vitamin C has auxiliary effects on the treatment of a variety of cancers.PTPN12 is a protein tyrosine phosphatase commonly expressed in mammalian tissues,which can affect cell migration and adhesion by regulating the tyrosine phosphorylation of various substrates.Recent studies have shown that PTPN12 is a potential therapeutic target for cancer,diabetes,metabolic diseases,autoimmune diseases and other diseases.We have proved that vitamin C can affect intracellular signal transduction pathways,including JAK/STAT,MAPK/ERK and other signal pathways through its receptor-like transporter SVCT2 in the form of signal molecules.In this study,we used HEK293 T cells and B16-F10 cells as target cells and found that vitamin C can promote PTPN12 serine phosphorylation through the MAPK/ERK signaling pathway,and then participate in the regulation of intracellular signaling pathways.At the same time,we studied the effects of different concentrations of vitamin C on the proliferation and migration of mouse melanoma cells through the MAPK/ERK signaling pathway,and studied the role of PTPN12 in the above process.The main research contents are as follows:1.Screening and verification of the downstream target protein PTPN12 in the Vitamin C-MAPK/ERK signaling pathway.First,through the analysis of phosphorylation quantitative proteomic data and the prediction of related targets,it is determined that PTPN12 may be one of the target proteins of the Vitamin C-MAPK/ERK signaling pathway.Then,combining with amino acid site mutation and Western Blot,we identified the specific phosphorylation sites on PTPN12 regulated by the vitamin C-MAPK/ERK signaling pathway as Ser434 and Ser632.And then,silver staining,phosphatase activity determination and other methods were used to verify that the phosphorylation of Ser434 and Ser632 on PTPN12 would inhibit PTPN12 phosphatase activity.2.Regulation of Vitamin C-MAPK/ERK-PTPN12 on JAK2 kinase.First,the co-localization of PTPN12 and JAK2 in the cell was confirmed by immunofluorescence staining and fluorescence resonance energy transfer.Then,combined with protein immunoprecipitation and Western Blot,it was verified that there is an interaction between PTPN12 and JAK2,and that PTPN12 can regulate the phosphorylation of tyrosine1007/1008 of JAK2.It has also been proved that vitamin C can inhibit the phosphatase activity of PTPN12 by up-regulating the phosphorylation of the serine site of PTPN12,thereby maintaining the activation of JAK2 kinase in the cell.3.The effect of vitamin C-MAPK/ERK-PTPN12 pathway on mouse melanoma cells.First,through Western Blot,it was verified that in B16-F10 cells,low concentration of vitamin C activates ERK1/2,while high concentration of vitamin C can inhibit ERK1/2continuously;then through wound healing assay and Transwell experiments,it was found that high concentrations of vitamins C inhibits cell migration in a a PTPN12-dependent manner,and low concentration vitamin C promotes cell migration;then,by constructing a B16-F10 cell line with PTPN12 knocked down,it was verified that the decrease of PTPN12 in B16-F10 cells would promote cell proliferation and migration,and lead to the increase of JAK2 activity.4.The effect of different concentrations of vitamin C on the growth of mice melanoma in situ.In this study,a tumor-bearing mouse model induced by B16-F10 cells was established,and it was verified that the lack of PTPN12 can promote the growth of mice melanoma in situ.Then by intra-tumoral injection of different concentrations of sodium ascorbate,we verified that low concentrations of sodium ascorbate promote the growth of in situ melanoma in mice,while high concentrations of sodium ascorbate have a significant inhibitory effect.In short,this study further proved the existence of the positive feedback regulation pathway of Vitamin C-JAK2-ERK-PTPN12-JAK2 on the basis of the previous research of our group,and for the first time found that different concentrations of Vitamin C can affects tumor cell proliferation,migration and other life activities through MAPK/ERK-PTPN12 signal pathway,and reveal the potential mechanism of high-concentration Vitamin C inhibiting mouse melanoma.In summary,on the basis of previous studies,this study proved that vitamin C promotes the serine phosphorylation of PTPN12-related sites by activating the MAPK/ERK signaling pathway,thereby inhibiting the dephosphorylation of JAK2 kinase by PTPN12 and maintaining the activation of JAK2 kinase,and playing a positive feedback regulatory role.We discovered for the first time that different concentrations of vitamin C can affect the migration of tumor cells in a PTPN12-dependent manner,and at the same time explored the potential mechanism of high concentrations of vitamin C in inhibiting skin melanoma in mice.